3rd Quarter 2015Compiled by Dr Ruchika Kohli

BRUCELLOSIS - UPDATE ON LABORATORY DIAGNOSIS

IntroductionHuman brucellosis is a zoonotic disease with a major impact on public health.

The number of undetected cases still remains high and this can be attributed to the non-specific clinical picture of human brucellosis, lowawareness of the disease in non-endemic countries and shortcomings in laboratory diagnosis.

The number of human cases is directly correlated with the number of infected animals within a defined region. Once the disease has beentransmitted from its animal reservoir to humans, only early diagnosis and adequate antibiotic therapy can prevent serious sequelae in patients.

Pathogenesis & PathologyThe common routes of infection in humans are the intestinal tract (ingestion of infected milk), mucous membranes (droplets), and skin(contact with infected tissues of animals). Cheese made from unpasteurised sheep or goats' milk is a particularly common vehicle.

The Brucella spp. that infect humans have apparent differences in pathogenicity:- B. abortus usually causes mild disease without suppurative complications; non-caseating granulomas of the reticulo-endothelial system are found. - B. canis also causes mild disease. - B. suis infection tends to be chronic with suppurative lesions; caseating granulomas may be present. - B. melitensis infection is more acute and severe.

Clinical FindingsThe incubation period is 1 � 6 weeks. The onset is insidious, with malaise, fever, weakness, arthralgia, and sweats. The fever usually rises in theafternoon, and falls during the night accompanied by drenching sweat. There may be gastrointestinal and nervous symptoms. Lymph nodesenlarge, and the spleen becomes palpable. Hepatitis may be accompanied by jaundice. Deep pain and disturbances of motion, particularly invertebral bodies, suggest osteomyelitis. These symptoms of generalised Brucella infection generally subside in weeks or months, althoughlocalised lesions and symptoms may continue.

Following the initial infection, a chronic stage may develop, characterized by weakness, aches and pains, low-grade fever, nervousness, andother non-specific symptoms compatible with neuropsychiatric abnormalities. Brucella spp. cannot be isolated from the patient at this stage,but the agglutination titre may be high.

The diagnosis of "chronic brucellosis" is difficult to establish with certainty unless local lesions are present.

Diagnostic Laboratory TestsCultureThe number of bacteria in clinical samples may vary widely, with the isolation of Brucella being highly dependent on the stage of disease(acute versus chronic), antibiotic pre-treatment, the existence of an appropriate clinical specimen and the culturing methods used. Isolationrates are much higher during the first two weeks of symptomatic disease and in blood or bone marrow cultures taken during the pyrexial phase.

SerologyIn contrast to bacterial culture, serological testing is fast, non-hazardous and more sensitive, and is therefore preferred in routine clinicalpractice. However, serological tests can only indirectly detect Brucella infections by high or rising titres of specific antibodies. Serological testscan also not be used for speciation due to considerable cross-reactivity between Brucella species. Cross-reaction between Brucella and othergram-negative bacteria (e.g. Yersinia enterocolitica, Francisella tularensis and Vibrio cholera) may lead to false-positive serology results.

Agglutination titres ≥ 1:160 or a fourfold rise in titre between acute and convalescent sera taken at least 14 days apart are considered to beindicative of active infection. However, diagnostic high titres can be detected months or even years after acute infection despite therapeuticsuccess and negative blood cultures. Patients in endemic regions may have persistent antibody titres due to ongoing exposure to Brucella.

Effective antibiotic therapy is usually accompanied by a decline in antibody titres, whereas persisting high IgG titres can be an indication oftreatment failure. Relapse is often characterised by a second peak of anti-Brucella IgG (but usually not IgM) immunoglobulins.

IgM antibody levels rise during the first week of acute illness, peak at 3 months, and may persist during chronic disease. Even with appropriateantibiotic therapy, high IgM levels may persist for up to 2 years in a small percentage of patients. IgG antibody levels rise about 3 weeks afteronset of acute disease, peak at 6 � 8 weeks, and can remain detectable lifelong despite successful treatment.

Molecular testingPolymerase chain reaction (PCR) assays can be used to detect Brucella DNA in pure cultures and in clinical specimens. Direct detection ofBrucella DNA in patient samples is a challenge because of the small number of bacteria present in clinical samples and inhibitory effects arisingfrom matrix components.

Challenges in laboratory testingIn endemic countries with limited financial means, diagnosis of brucellosis should rely on two criteria: clinical presentation and laboratorydiagnosis. This diagnosis should be based either on a positive culture from blood or organ samples or on positive serological results, preferablydemonstrating rising titres. In rare cases, clinical diagnosis without laboratory confirmation can justify therapy.

TreatmentBrucella spp. may be susceptible to tetracyclines or ampicillin. Symptomatic relief usually occur within a few days after treatment with thesedrugs is begun. However, because of their intracellular location, the organisms are not readily eradicated completely from the host. For bestresults, treatment must be prolonged. Combined treatment with a tetracycline (such as doxycycline) for 6 weeks PLUS either streptomycin for2 � 3 weeks or rifampicin for 6 weeks is recommended.

Epidemiology, Prevention, & ControlBecause of occupational exposure (e.g. farmer and veterinarian), Brucella infection is much more frequent in men.The majority of infections remain asymptomatic (latent).

Active immunisation of humans against Brucella infection is experimental only. Control rests on limitation of spread and possible eradication ofanimal infection, pasteurisation of milk and milk products, and reduction of occupational hazards wherever possible.

References1. Jawetz, Melnick, & Adelberg's Medical Microbiology; 24th edition, 2007;2. Al Dahouk S, et al. New developments in the diagnostic procedures for zoonotic brucellosis in humans; Rev Sci Tech 2013; 32: 177 � 188.

Corporate branding/newsletters/south africa/2015/south africa - brucellosis - update on laboratory diagnosis may2015.cdrdesign done and printed by: ELECTRONIC LABORATORY SERVICES (PTY) LTD 0027 (0) 11 358-0798/99

www.lancet.co.za @LancetLab

HEAD OFFICELancet Corner, Ground Floor, Cnr Menton and Stanley Roads, Richmond, Johannesburg, South Africa. (011) 358 0800

PRETORIA MAIN LABORATORY1st Floor Pencardia Building, 509 Pretorius Street, Arcadia, South Africa. (012) 483 0100

DURBAN MAIN LABORATORY 102 Lancet Medical Centre, 74 Ismail C Meer Street, Durban, South Africa. (031) 308 6500